title: Cytochrome c folding traps are not due solely to histidine-heme ligation: direct demonstration of a role for N-terminal amino group-heme ligation.
abstract: In previous work, heme ligation effects on the folding of cytochrome c have been attributed to histidine side-chains. A variant of yeast iso-1-cytochrome c designated TM, which lacks all histidine residues except His18, still shows evidence of denatured state heme ligation in the pH range between 5 and 6 where normally only histidine ligation is expected. Conversion of the N-terminal amino group of TM to a carbonyl group through a transamination reaction with glyoxylate produced a protein (ModTM) with no terminal amino group. The midpoint pH (pH1/2) for loss of heme ligation in 3 M guanidine-HCl shifts from 5.9 to 7.4 as a result of this modification, providing direct evidence for N-terminal amino group-heme ligation under these conditions. The N-terminal amino group thus competes with histidine for misligation of iso-1-cytochrome c under denaturing conditions. To assess the effect of denatured state N-terminal amino group-heme ligation on the folding of iso-1-cytochrome c, stopped-flow kinetics experiments were conducted. At pH 6.2, the major refolding lifetimes (3 M-->0.27 M guanidine-HCl) for ModTM, TM and the wild-type protein are 11.6 ms, 30 ms and 1.3 seconds, respectively. Denatured state ligation of the N-terminal amino group thus slows folding 2.6-fold.
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, 2190 East Iliff Avenue, Denver, CO, 80208-2436, USA.
authors.forename: B
authors.initials: B
authors.lastname: Hammack
authors.forename: S
authors.initials: S
authors.lastname: Godbole
authors.forename: B E
authors.initials: BE
authors.lastname: Bowler
mesh_terms: D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D003574:Cytochrome c Group; D006418:Heme; D006639:Histidine; D019791:Guanidine